Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli 0157

BRITISH STANDARD BS EN ISO 16654:2001 Incorporating Corrigendum No. 1 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli 0157 The European Standard EN ISO 16654:2001 has the status of a British Standard ICS 07.100.30 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW

BS EN ISO 16654:2001 National foreword This British Standard is the official English language version of EN ISO 16654:2001. It is identical with ISO 16654:2001. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/european committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications may be found in the BSI Standards Catalogue under the section entitled International Standards Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to v, a blank page, pages 1 to 13, the annex ZA page, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 August 2001 Amendments issued since publication Amd. No. Date Comments 13344 Corrigendum No. 1 July 2001 Incorporation of annex ZA BSI 07-2001 ISBN 0 580 37719 9

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM EN ISO 16654 May 2001 ICS 07.100.30 English version Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O 157 (ISO 16654:2001) Microbiologie des aliments - Méthode horizontale pour la recherche des Escherichia coli O 157 (ISO 16654:2001) Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den Nachweis von Escherichia coli O 157 (ISO 16654:2001) This European Standard was approved by CEN on 1 May 2001. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16654:2001 E

Foreword The text of the International Standard ISO 16654:2001 has been prepared by Technical Committee ISO/TC 34 "Agricultural food products" in collaboration with Technical Committee CEN/TC 275 "Food analysis - Horizontal methods", the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2001, and conflicting national standards shall be withdrawn at the latest by November 2001. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard ISO 16654:2001 was approved by CEN as a European Standard without any modification. NOTE: Normative references to International Standards are listed in annex ZA (normative).

INTERNATIONAL STANDARD EN ISO 16654:2001 ISO 16654 First edition 2001-05-01 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 Microbiologie des aliments Méthode horizontale pour la recherche des Escherichia coli O157 Reference number ISO 16654:2001(E)

Contents Page Foreword...iv Introduction...v 1 Scope...1 2 Normative references...1 3 Term and definition...1 4 Principle...2 5 Culture media, reagents and antisera...2 6 Apparatus and glassware...7 7 Sampling...8 8 Preparation of test sample...8 9 Procedure (see annex A)...8 9.1 Test portion and initial suspension...8 9.2 Enrichment...8 9.3 Immunomagnetic separation (IMS)...8 9.4 Plating out onto selective agars and identification of E. coli O157 colonies...9 9.5 Confirmation...10 9.6 Further characterization...11 10 Quality assurance...11 10.1 Test strains for quality assurance purposes...11 10.2 Culture method...11 11 Expression of results...11 12 Test report...11 Annex A (normative) Diagram of procedure...12 Bibliography...13 Annex ZA (normative) Normative references to international publications with their relevant European publications...14 iii

Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 16654 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. Annex A forms a normative part of this International Standard. iv

Introduction Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain products. In this case, different methods specific to these products may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this horizontal method as far as possible. When this International Standard is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of particular products. The harmonization of test methods cannot be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with this horizontal method. It is hoped that when such standards are reviewed they will be changed to comply with this International Standard so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons. v

Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 WARNING Escherichia coli O157 can cause severe life-threatening illness and has a low infective dose. Laboratory-acquired infections have been reported. In order to safeguard the health of laboratory personnel, it is essential that the whole of this method be carried out only by skilled personnel using good laboratory practices and preferably working in a containment facility. Relevant national Health and Safety Regulations relating to this organism must be adhered to. Care must be taken in the disposal of all infectious materials. 1 Scope This International Standard specifies a horizontal method for the detection of Escherichia coli serogroup O157. Subject to the limitations discussed in the introduction, this International Standard is applicable to products intended for human consumption or for animal feeding stuffs. 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions. ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Term and definition For the purposes of this International Standard, the following term and definition applies. 3.1 Escherichia coli O157 E. coli O157 microorganisms which form typical colonies on the surface of the plating-out medium used in this International Standard, and which produce indole and agglutinate specifically with antiserum against the O157 antigen NOTE 1 NOTE 2 Sorbitol-positive E. coli O157 strains are not detected on CT-SMAC (5.2) media. Some indole-negative mutations have been found. 1

4 Principle The detection of Escherichia coli O157 necessitates four successive stages (see annex A). a) Enrichment of the test portion homogenized in modified tryptone soya broth containing novobiocin (mtsb + N) with incubation at 41,5 C 1 C for 6 h and subsequently for a further 12 h to 18h. b) Separation and concentration of microorganisms by means of immunomagnetic particles coated with antibodies to E. coli O157. c) Isolation by subculture of the immunomagnetic particles with adhering bacteria onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC) and the user's choice of a second selective isolation agar. d) Confirmation of sorbitol-negative colonies from CT-SMAC and colonies typical of E. coli O157 on the second isolation agar, by indole production and agglutination with E. coli O157 antiserum. NOTE Further characterization, by for example pathogenic markers, of the positive isolates can be obtained by forwarding them to an appropriate reference laboratory. 5 Culture media, reagents and antisera For current laboratory practices, see ISO 7218. 5.1 Enrichment medium: Modified tryptone soya broth with novobiocin (mtsb + N) See reference [1]. 5.1.1 Modified tryptone soya broth (mtsb) 5.1.1.1 Composition Enzymatic digest of casein Enzymatic digest of soya D(+)-glucose Bile salts No. 3 Sodium chloride Dipotassium hydrogen phosphate (K 2 HPO 4 ) Water 17,0 g 3,0 g 2,5 g 1,5 g 5,0 g 4,0 g 1 000 ml 5.1.1.2 Preparation Dissolve the components or the dehydrated complete medium in the water, by heating if necessary. Adjust the ph, using the ph-meter (6.6), if necessary, so that after sterilization it is 7,4 0,2 at 25 C. Dispense the medium in appropriate amounts in flasks or bottles (6.7). Sterilize for 15 min in the autoclave (6.1) set at 121 C. 2

5.1.2 Novobiocin solution 5.1.2.1 Composition Novobiocin Water 5.1.2.2 Preparation 0,45 g 100 ml Dissolve the novobiocin in the water and sterilize by membrane filtration. Prepare on the day of use. 5.1.2.3 Preparation of the complete medium Immediately before use, add 1 ml or 4 ml of novobiocin solution (5.1.2) to either 225 ml or 900 ml of cooled mtsb (5.1.1). The final concentration of novobiocin is 20 mg per litre of mtsb. 5.2 First selective isolation medium: Cefixime tellurite sorbitol MacConkey agar (CT-SMAC) See reference [2]. 5.2.1 Base medium 5.2.1.1 Composition Enzymatic digest of casein Enzymatic digest of animal tissues Sorbitol Bile salts No. 3 Sodium chloride Neutral Red Crystal Violet Agar Water a Depending on the gel strength of the agar. 17,0 g 3,0 g 10,0 g 1,5 g 5,0 g 0,03 g 0,001 g 9gto18g a 1 000 ml 5.2.1.2 Preparation Dissolve the basic components or the complete dehydrated base in the water by boiling. Adjust the ph (6.6), if necessary, so that after sterilization it is 7,1 0,2 at 25 C. Sterilize for 15 min in the autoclave (6.1) set at 121 C. 5.2.2 Potassium tellurite solution 5.2.2.1 Composition Potassium tellurite for bacteriological use Water 0,25 g 100 ml 3

5.2.2.2 Preparation Dissolve the potassium tellurite in the water and sterilize by membrane filtration. This solution may be stored at room temperature for up to 1 month, but discard it if a white precipitate forms. 5.2.3 Cefixime solution 5.2.3.1 Composition Cefixime Water 5,0 mg 100,0 ml 5.2.3.2 Preparation Dissolve the cefixime in the water and sterilize by membrane filtration. NOTE Cefixime may need to be dissolved in ethanol. This solution may be stored at 3 C 2 C for 1 week. 5.2.4 Complete medium 5.2.4.1 Composition Base medium (5.2.1) Potassium tellurite solution (5.2.2) Cefixime solution (5.2.3) 1 000 ml 1,0 ml 1,0 ml 5.2.4.2 Preparation Either cool the freshly sterilized base medium (5.2.1) to between 44 C and 47 C (6.5), or melt it by steaming the previously sterilized and solidified base medium, then cool to between 44 C and 47 C. Add 1 ml of the tellurite solution and 1 ml of the cefixime solution to 1000 ml of the base medium. Mix and pour about 15 ml amounts into sterile Petri dishes (6.15). Allow to solidify. The final concentration of tellurite is 2,5 mg/l and cefixime 0,05 mg/l. Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facing downwards, in an oven set at a temperature between 25 C and 50 C (6.2), until the droplets have disappeared from the surface of the medium. Do not dry them any further. The agar plates may also be dried in a laminar-flow safety cabinet for 30 min with half-open lids, or overnight with the lids in place. If prepared in advance, the undried plates may be stored in the dark in plastic bags or other moisture-retentive containers, in a refrigerator at 3 C 2 C for up to 2 weeks. 5.3 Second selective isolation medium Use any other solid selective medium, at the choice of the laboratory, complementary to CT-SMAC agar and especially appropriate for the isolation of Escherichia coli O157. Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facing downwards, in an oven set at a temperature between 25 C and 50 C (6.2), until the droplets have disappeared 4

from the surface of the medium. Do not dry them any further. The agar plates may also be dried in a laminar-flow safety cabinet for 30 min with half-open lids, or overnight with the lids in place. If prepared in advance, the undried plates may be stored in the dark in plastic bags or other moisture-retentive containers, in a refrigerator at 3 C 2 C for a time that causes no change to its performance. 5.4 Nutrient agar 5.4.1 Composition Meat extract Peptone Agar Water a Depending on the gel strength of the agar. 3,0 g 5,0 g 9gto18g a 1 000 ml 5.4.2 Preparation Dissolve the components or the dehydrated complete medium in the water, by heating if necessary. Adjust the ph, if necessary, so that after sterilization it is 7,0 0,2 at 25 C. Transfer the medium into flasks or bottles (6.7) of appropriate capacity. Sterilize for 15 min in the autoclave (6.1) set at 121 C. 5.4.3 Preparation of nutrient agar plates Transfer about 15 ml of the molten, cooled medium (5.4.2) at between 44 C and 47 C (6.5) to Petri dishes and allow to solidify. Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facing downwards, in an oven set at a temperature between 25 C and 50 C (6.2), until the droplets have disappeared from the surface of the medium. Do not dry them any further. The agar plates may also be dried in a laminar-flow safety cabinet for 30 min with half-open lids, or overnight with the lids in place. If prepared in advance, the undried plates may be stored in the dark, in plastic bags or other moisture-retentive containers, in a refrigerator at 3 C 2 C for up to 2 weeks. 5.5 Tryptone/tryptophan medium 5.5.1 Composition Tryptone Sodium chloride DL-Tryptophan Water 10,0 g 5,0 g 1,0 g 1 000 ml 5.5.2 Preparation Dissolve the components in the water by boiling if necessary. Adjust the ph (6.6) so that after sterilization it is 7,5 0,2 at 25 C. 5

Dispense in 5 ml amounts into test tubes or bottles (6.7) of appropriate capacity. Sterilize for 15 min in the autoclave (6.1) set at 121 C. 5.6 Kovac s indole reagent 5.6.1 Composition 4-Dimethylaminobenzaldehyde 2-Methylbutan-1-ol or pentan-1-ol Hydrochloric acid (H 20 1,18 g/ml to 1,19 g/ml) 5,0 g 75,0 ml 25,0 ml 5.6.2 Preparation Dissolve the 4-dimethylaminobenzaldehyde in the alcohol, by warming if necessary in a water bath (6.5) maintained at between 44 C and 47 C. Cool to room temperature and add the hydrochloric acid. Protect from light in a brown glass bottle and store at 3 C 2 C. The reagent shall be light yellow to light brown and free of precipitate. 5.7 Anti-Escherichia coli O157 immunomagnetic particles These are immunomagnetic particles coated with specific antibodies against E. coli O157 for concentration and separation of these microorganisms. NOTE They are available from commercial sources. The manufacturer s instructions should be followed precisely regarding their preparation for use. 5.8 Wash buffer: Modified phosphate buffer, 0,01mol/l, of ph 7,2 5.8.1 Composition Sodium chloride Potassium chloride Disodium hydrogen phosphate (anhydrous) Potassium dihydrogen phosphate (anhydrous) Polyoxyethylene sorbitan monolaurate (Tween 20 syrup) Water 8,0 g 0,2 g 1,44 g 0,24 g 0,2 ml 1 000 ml 5.8.2 Preparation Dissolve the components in water. Adjust the ph (6.6), if necessary, to 7,2 0,2 at 25 C. Dispense in bottles or flasks (6.7) in appropriate volumes for use Sterilize for 15 min in the autoclave (6.1) set at 121 C. The solution may appear cloudy but becomes clear on standing. Commercially available phosphate buffer with the same composition and the same performance may be used. 6

5.9 Normal saline solution 5.9.1 Composition Sodium chloride Water 8,5 g 1 000 ml 5.9.2 Preparation Dissolve the sodium chloride in the water. Dispense in bottles or flasks (6.7) in appropriate volumes for use. Sterilize for 15 min in the autoclave (6.1) set at 121 C. 5.10 Escherichia coli O157 antiserum, available either from specialist laboratories or from commercial sources as separate somatic "O" 157. The antiserum shall be tested with positive and negative controls prior to use on unknown isolates. 6 Apparatus and glassware Usual microbiological equipment (see ISO 7218) and, in particular, the following. 6.1 Apparatus for dry sterilization (oven) and/or wet sterilization (autoclave) See ISO 7218. 6.2 Drying cabinet or incubator, capable of being maintained between 25 C and 50 C. 6.3 Incubator, capable of being maintained at 37 C 1 C. 6.4 Incubator, capable of being maintained at 41,5 C 1 C. 6.5 Water bath, capable of being maintained at between 44 C and 47 C. 6.6 ph-meter, capable of being read to the nearest 0,01 ph unit at 25 C, enabling measurements to be made which are accurate to 0,1 ph unit. 6.7 Test tubes, flasks or bottles, of appropriate capacity, for sterilization and storage of culture media and incubation of liquid media. 6.8 Measuring cylinders, of appropriate capacity, for preparation of dilutions and complete media. 6.9 Total-delivery graduated pipettes, of nominal capacities 1 ml and 10 ml, graduated in 0,1 ml and 0,5 ml divisions, respectively. 6.10 Loops and wires, made of platinum/iridium or nickel/chrome or Pasteur pipettes or single-use loops. 6.11 Mechanical air-displaced pipettors, sterile, with an operating range from 20 l to 200 l with 10 l divisions, or similar. 6.12 Magnetic separator with magnetic rack, for concentration of immunomagnetic particles, for use with Eppendorf-type plastic tubes (6.13). 6.13 Eppendorf-type plastic tubes, with screw caps, sterile, disposable, centrifuge type, of 1,5 ml capacity to fit the magnetic rack. 7

Avoid the creation of aerosols when opening. 6.14 Rotary mixer (windmill type, blood sample mixer), capable of rotating at 15 r/min to 20 r/min. 6.15 Petri dishes, of diameter 90 mm and 140 mm. 6.16 Vortex mixer 7 Sampling It is important that the laboratory receive a sample which is truly representative and that has not been damaged or changed during transport or storage. It is recommended to cool the sample quickly before storage. Sampling is not part of the method specified in this International Standard. If there is no specific International Standard dealing with sampling the product concerned, it is recommended that the parties concerned come to an agreement on this subject. 8 Preparation of test sample Prepare the test sample in accordance with the specific International Standard appropriate to the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject. 9 Procedure (see annex A) 9.1 Test portion and initial suspension See ISO 6887-1 and any specific International Standard appropriate to the product concerned. NOTE Further parts of ISO 6887 are in preparation, see bibliography. In general, to prepare the initial suspension, add a test portion of x gorx ml to 9x ml or 9x g of modified tryptone soya broth with novobiocin (mtsb + N) (5.1), pre-warmed in the incubator (6.4) to 41,5 C to obtain a ratio of test portion to mtsb + N of 1/10 (mass to volume, or volume to volume). It is recommended to use stomacher bags with mesh inserts to reduce the interference of food particles with immunocapture kits (9.3). 9.2 Enrichment Incubate (6.4) the initial suspension, prepared in accordance with 9.1, at 41,5 C for 6 h, and subsequently for a further12hto18h(i.e.toatotalelapsedtimeof18hto24h). A 6-h incubation followed by immunomagnetic separation and plating onto selective agars can yield a presumptive positive result which can become negative after a further 18-h incubation. 9.3 Immunomagnetic separation (IMS) 9.3.1 General IMS should be carried out after 6 h and again, if necessary, after 12 h to 18 h of incubation. 8

The instructions below are for general guidance and may not be complete in all details. Therefore the manufacturer's instructions should be followed concerning the procedure and method for the use of immunocapture kits and the equipment needed. 9.3.2 Immunocapture WARNING Use aseptic techniques to avoid any external contamination and the creation of aerosols. Perform this protocol in a containment safety cabinet, if available. Wear gloves. Using the magnetic separator (6.12) and antibody-coated immunomagnetic particles (5.7), carry out the following capture/separation procedure. Mix the enrichment culture (9.2) and allow any coarse food materials to settle out. To an Eppendorf-type plastic tube (6.13), add 20 l of the prepared immunomagnetic particles (5.7) at room temperature. Take 1 ml of the upper liquid from the enrichment culture, avoiding if possible the transfer of any food particles or fatty materials, and transfer to the Eppendorf-type plastic tube. Mix the suspension on the rotary mixer (6.14) set at about 12 r/min to 20 r/min for 10 min. 9.3.3 Separation Place each Eppendorf-type plastic tube (see 9.3.2) in the magnetic rack (6.12) and allow the magnetic particles to congregate against the magnet by gently rocking the rack through 180. Open the cap carefully without disturbing the particles on the wall of the tube. Using a new sterile Pasteur pipette (6.10) for each sample and with the tube still in the magnetic rack, remove the liquid by sucking slowly from the bottom of the tube. Add 1 ml of sterile wash buffer (5.8) and replace the cap. Remove the magnet from the rack. Mix the contents of the tubes by gentle inversion of the rack through 180 and then return the magnet to the rack. Take care to avoid cross contamination when adding fresh buffer. Proceed as above to remove the wash liquid with a new Pasteur pipette for each sample. Repeat the washing procedure several times. Remove from the magnetic separator and add 100 l of sterile wash buffer (5.8) to the tube and re-suspend the magnetic particles. NOTE This procedure could be difficult to apply to fat products or fresh cheese. 9.4 Plating out onto selective agars and identification of E. coli O157 colonies 9.4.1 Plating out Using a mechanical-type pipettor (6.11), transfer 50 l of the washed and re-suspended magnetic particles (9.3.3) to a pre-dried plate of cefixime tellurite sorbitol MacConkey agar (5.2) and also 50 l to a pre-dried plate of the second isolation medium (5.3). Streak out the particles using a sterile loop (6.10) to obtain many well-isolated colonies over the agar. Incubate (6.3) the CT-SMAC (5.2) at 37 C for 18 h to 24 h, and incubate the second selective agar at its recommended temperature and specified time. Depending on the type of food sample and its microbial flora, incubation of the enrichment broth for 20 h to 24 h may give rise to a heavy growth of other bacteria on the selective agar plates so that colonies of E. coli O157 are difficult to find. Inoculation of selective agars with dilutions of the IMS preparation or volumes less than 50 l per plate can increase the chance of gaining separated colonies of E. coli O157 but note that this can increase the detection limit as well. 9

9.4.2 Recognition of typical E. coli O157 colonies On CT-SMAC agar, typical colonies are transparent and almost colourless with a pale yellowish-brown appearance and a diameter of approximately 1 mm. Examine the second selective isolation agar for typical colonies of E. coli O157 following the manufacturer's instructions. 9.5 Confirmation NOTE Commercially available miniaturized biochemical identification kits that permit the identification of sorbitol-negative and indole-positive E. coli and latex agglutination kits for E. coli O157 may be used, provided appropriate tests with known positive and negative strains are carried out to confirm performance. 9.5.1 Selection of colonies Take five typical colonies from each plate, as selected in 9.4. If an agar plate contains less than 5 typical colonies, all the colonies shall be examined. Streak each selected colony onto a plate of nutrient agar (5.4) to allow well-separated colonies to develop. Incubate (6.3) the plates for 18 h to 24 h at 37 C. Use only pure cultures from the nutrient agar plate for the tests described in 9.5.2 and 9.5.3. 9.5.2 Biochemical confirmation: Indole formation Inoculate one colony from the pure culture on nutrient agar (9.5.1) into a tube of tryptone/tryptophan medium (5.5). Incubate (6.3) at 37 C for 24 h. Add 1 ml of Kovac's reagent (5.6) and allow to stand at room temperature for 10 min. The formation of a red colour indicates a positive reaction. A yellow/brown colour indicates a negative reaction. 9.5.3 Serological identification 9.5.3.1 General Only examine indole-positive colonies for their serological reaction with antiserum to E. coli O157. 9.5.3.2 Elimination of auto-agglutinating isolates Place a drop of saline solution (5.9) onto a cleaned glass slide. Using a loop (6.10), mix into this drop one colony from the nutrient agar plate (9.5.1) so as to obtain a homogeneous and turbid suspension. Rock the slide gently for 30 s to 60 s. Observe the result against a dark background and, if necessary, with the aid of a magnifying lens. If the suspension has formed visible clumps, the strain is considered to auto-agglutinate and shall not be tested further, as the reaction with the specific antiserum is not possible. 10

9.5.3.3 Reaction with E. coli O157 antiserum Using a pure colony from the nutrient agar (9.5.1), suspend it in a fresh drop of saline as in 9.5.3.2 and add a small drop of E. coli O157 antiserum (5.10). If agglutination occurs within 1 min, the reaction is positive. 9.5.3.4 Positive identification Consider as positive isolates those that are indole positive and react with either O157 antiserum or O157 plus H7 antisera, if available. 9.6 Further characterization For further identification of positive colonies for the detection of flagellar antigens and for pathogenic characteristics, cultures should be sent to a Reference Laboratory. 10 Quality assurance 10.1 Test strains for quality assurance purposes Strains of E. coli O157 that do not carry the virulence factors attributed to pathogenicity are available from national or international culture collections. These are recommended for quality assurance testing of media and antisera. 10.2 Culture method To check the ability of the laboratory and media to detect low numbers of Escherichia coli O157 in the food samples under test by the method described in this International Standard, reference samples of a low inoculum of a non-pathogenic E. coli O157 and a large inoculum of another strain of E. coli should be run in parallel with the test sample. 11 Expression of results In accordance with the interpretation of the results, report the presence or absence of Escherichia coli O157 in the test portion, specifying the mass in grams or the volume in millilitres of the sample tested. 12 Test report The test report shall specify the following: a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used, with reference to this International Standard; d) the temperature of incubation; e) all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the results; f) the test results obtained. The test report shall also state if further tests are to be carried out by a reference laboratory or, if done, what the results were. 11

Annex A (normative) Diagram of procedure Test portion (x gorx ml) 9 x ml mtsb + N (5.1) pre-warmed to 41,5 C (9.1) Homogenization and incubation for 6 h then a further 12 h to 18 h at 41,5 C (9.2) Concentration of E. coli O157 by capture onto immunomagnetic particles and washing with sterile wash buffer (9.3) Re-suspension in 0,1 ml of sterile wash buffer (9.3) Inoculation of 50 l of the washed and re-suspended magnetic particles on selective medium to obtain isolated colonies (9.4) CT-SMAC medium (5.2) Second isolation medium (5.3) Incubation at 37 C for 18 h to 24 h Taking five typical sorbitol-negative colonies Incubation at its recommended temperature and specified time Taking five typical colonies Confirmation of pure colonies (9.5.1) by indole formation (9.5.2) or by commercially available biochemical identification kits (9.5) and by serological identification with antiserum to E. coli O157 (9.5.3) For further identification, cultures should be sent to a reference laboratory (9.6) 12

Bibliography [1] DOYLE M.P. and SCHOENI J.L. Appl. Environ. Microbiol., 53, 1987, pp. 2394-2396. [2] ZADIK P.M., CHAPMAN P.A. and SIDDONS C.A. J. Med. Microbiol., 39, 1993, pp. 155-158. [3] ISO 6887-2, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 2: Specific rules for the preparation of the test samples and initial suspension of meat and meat products. [4] ISO 6887-3, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 3: Specific rules for the preparation of the test samples and initial suspension of milk and milk products. [5] ISO 6887-4, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 4: Specific rules for the preparation of the test samples and initial suspension of fish products. [6] ISO 6887-5, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 5: Specific rules for the preparation of the test samples and initial suspension of products other than milk and milk products, meat and meat products and fish products. 13

Annex ZA (normative) Normative references to international publications with their relevant European publications This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies (including amendments). NOTE Where an International Publication has been modified by common modifications, indicated by (mod.), the relevant EN/HD applies. Publication Year Title EN Year ISO 6887-1 1999 Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 1: General rules for the preparation of the initial suspension and decimal dilutions EN ISO 6887-1 1999.14

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